Fluorescence Gets Easier to Measure

BALTIMORE -- Measurements of fluorescence lifetimes provide important information about fluorophores that is not available from simple intensity measurements. Lifetimes in the nanosecond range typically are determined by observing the decrease in fluorescence intensity following a pulse of excitation radiation, or by frequency-domain methods. Until very recently, these methods required expensive research-grade equipment. A new generation of digital signal processor lock-in amplifiers now allows the determination of these short lifetimes with a relatively inexpensive, versatile setup.

Researchers led by Govind Rao at the University of Maryland Baltimore County and the University of Maryland Biotechnology Institute described their experimental setup in the February 1999 issue of Review of Scientific Instruments. Their work was supported by a National Institutes of Health grant and funding from Genentech, Merck and Pfizer.

The computer-controlled system included an SR844 RF lock-in amplifier from Stanford Research Systems of Sunnyvale, Calif., that can measure up to 200 MHz. A low-cost blue light-emitting diode provided the excitation source, and an R928 photomultiplier from Hama-matsu Corp. of Bridgewater, N.J., measured the emission. The lock-in amplifier modulated the intensity of the LED.
Researchers at the University of Maryland Baltimore County and its Biotechnology Institute are using a lock-in amplifier to determine fluorescence lifetimes. Here, blue light passes through the fiber into a sample of fluorescein, which is emitting green fluorescence. Part of the fluorescence goes back into the fiber to the detector. The lock-in amplifier analyzes the fluorescence to determine the sample's fluorescence lifetime.
The frequency domain technique, also referred to as phase modulation fluorometry, has been around for many years. When the intensity of the excitation light is modulated, the intensity of the emission is modulated at the same frequency, but it is shifted in phase. This phase shift can be related to the fluorescence lifetime. A modulation frequency of 100 MHz is required to determine lifetimes in the nanosecond range, and, until the digital signal processor became available, lock-in amplifiers that operated at these higher frequencies cost more than $100,000.

"The practical limit in the response frequency was between 80 MHz and 100 MHz," said researcher Peter Harms. "The limiting factor was the response rate of the blue LED that we used. It is difficult to modulate these devices quickly." He said that higher modulating frequencies could be obtained by using one of the new violet laser diodes in place of the LED. However, the advantage of this system is its approximately $10,000 price tag and its flexibility and ease of operation.

Harms said that this system is a good entry-level research tool. Besides its low cost, it is small enough to provide lifetime measurements on working industrial equipment. "Our lab uses lifetimes for chemical sensors, and this instrument allows us to determine the optimum frequency for each sensing chemistry. Commercial sensor systems will be even lower-cost if they are built around a single frequency," he said.